Epithelial polarity is key to the function of adult epithelial organs (kidney, liver, intestine and exocrine glands) and is fundamental for embryo organogenesis; its early loss is characteristic of many human carcinomas. The goal of this proposal is to study the cellular and molecular basis of the polarized surface distribution of plasma membrane proteins in epithelial cells. The sorting of viral envelope glycoproteins which are targeted to the apical surface (influenza hemagglutinin -HA) or to the basolateral surface (Vesicular Stomatitis Virus G protein -VSV G) in the Trans Golgi Network (TGN) of Madin Darby canine kidney (MDCK) cells will be studied by laser confocal scanning microscopy (LSCM). LSCM will also be used to study the role of the cytoskeleton in the exocytosis of VAC, a vacuolar apical compartment first described by our laboratory which plays an important role in the formation of the apical surface of developing epithelial monolayers, both in vivo and in vitro. Novel biotin polarity and targeting assays recently developed by this laboratory will be used to study the role of microtubules, glycolipids and epithelial tissue- type in the sorting and targeting of viral and endogenous plasma membrane glycoproteins in MDCK cells and in intestinal cell lines. The secretory and membrane default pathways of different epithelial types (kidney, intestine, thyroid, pancreas) will be characterized by transfecting cDNAs encoding for a variety of secretory proteins and for ER and lysosomal membrane proteins that, either by mutation or by changes in the culture conditions, are expressed in the plasma membrane. A chimeric-protein and transfection approach will be used to search for targeting information in transmembrane and cytoplasmic domains of uvomorulin (a basolateral protein) and Nerve Growth Factor Receptor (an apical protein). Permeabilized cell, genetic (mutant), and crosslinking approaches will be used to search for proteins, factors and metabolic requirements involved in the sorting and vectorial targeting of plasma membrane proteins. A megadalton apical complex recently identified by our laboratory will be characterized and its role in the development of the apical surface defined using methods recently used to study a basolateral fodrin-ankyrin-Na,K-ATPase-uvomorulin complex in MDCK cells. It is expected that these studies will contribute fundamental information to the process of epithelial cell organization and differentiation.